The primary hippocampal neurons, after dissociation from the hippocampal tissues of E18 Sprague-Dawley rat pups, were seeded on a poly-D-lysine-coated glass coverslip at density of 100 cells/mm² in the Neurobasal medium, which was replaced with the medium containing CBD (0.1–100 μM) at 1 DIV. In the case of 1% DMSO, which was the DMSO concentration for preparation of 100-μM CBD solution, we did not observe any noticeable difference compared with the nontreated control in cell viability and morphology. Throughout this article, we used the value of %viability, which was defined as % of cell viability with the nontreated control as a reference (i.e., the observed viability divided by the viability of the nontreated cells). After incubation for 24 h (i.e., at 2 DIV), the cells were stained with calcein AM for live cells (green) and ethidium homodimer-1 for dead cells (red), and the confocal laser-scanning microscope (CLSM) images were taken for the viability analysis.

The CLSM images (Fig. 1a) arguably showed the concentration-dependent neurotoxicity of CBD in the hippocampal neuron culture. For example, while the cell death was not noticeable up to 5 μM of CBD, the red fluorescence in CLSM images indicated that most hippocampal neurons were dead with 30 μM of CBD. The quantitative analysis showed that the %viability decreased to 44.80% with 10 μM of CBD, and the lethal concentration 50 (LC₅₀) value, the concentration at which the %viability was 50%, was calculated to be 9.85 μM (Fig. 1b). It is to be noted that this result is the first quantitative in vitro data, affording the LC₅₀ value of CBD, for the hippocampal-neuron culture.

The careful analysis of the CLSM images suggested that CBD might disrupt the cell adhesion. Some CLSM images had green-stained vestiges, and some dying cells, stained in both red and green, had partially detached neurites and cell body. We could frequently find the costained neurons with partial detachment as well as live neurons with no or few neurites, especially in the case of 10 μM of CBD.

To get some insights into the death-related cell detachment, the cells that remained attached to the culture plate were stained, at 2 DIV, with phalloidin for F-actin (green), CellTracker Red CMTPX Dye for cytoplasm (red), and DAPI for nuclei (blue). The CLSM images (Fig. 2) clearly showed that the neurons did not develop properly in the CBD-containing medium ([CBD]=10 μM), although the quantitative analysis was not feasible due to the cell detachment.

Although the developmental rate, judged by the Banker's classification of developmental stages,³⁴ seemed not to be affected by CBD, it was apparent that the neurons presented few, if not no, filopodia and lamellipodia structures. For example, no axonal branches were observed, and the growth cones were shrunken in size noticeably. In addition, we observed some cellular debris, such as only nuclei (top image in Fig. 2), condensed chromatin, and ruptured cells (middle image in Fig. 2). Based on the observation, it was hypothesized that the neuronal death by CBD might be considered to be a kind of detachment-induced apoptosis/anoikis (i.e., apoptosis following the loss of cell anchorage, especially the loss of cell/matrix interaction).^35,36

Detailed elucidation on the neurotoxic mechanism of CBD remains to be seen at this moment, but our results implied that the lipophilic CBD might interfere with the integrity of cell membranes, causing the cell rupture and death, at its high concentration. However, it is also to mention that our neurotoxicity results are not translated directly to the in vivo conditions. The brain is composed of neuronal and non-neuronal cells and others, and the in vivo tolerance of hippocampal neurons to CBD would be different from the in vitro conditions. For example, glial cells, such as astrocytes, play supporting roles in the brain. Relevant reports showed that the in vitro neurotoxicity of unnatural monosaccharides to primary hippocampal neurons was mitigated when a hippocampal tissue was used^26,28 or a neuron/astrocyte coculture was employed.²⁷ In this respect, it could be explained that the coculture system of neuron and glia (2:1) did not show the neurotoxicity of CBD.^10–12

It is to note that the H₂O₂ neurotoxicity experiments should be performed with caution under the tightly controlled conditions to ensure the data reproducibility, because of the rapid decomposition of H₂O₂ at ambient temperature. Specifically, the H₂O₂ stock solution (0.1 or 1 mM) was to be prepared at the low temperature (4°C) and transferred to the culture medium right after preparation (within 5 min). It was also found to be critical that the dilution of the H₂O₂ stock solution in the culture medium was done at 4°C, not at ambient temperature.

After 24 h of incubation at 37°C, the %viability was obtained by the analysis of the CLSM images (Fig. 3a). There was no significant difference in viability, compared with the control, up to 1 μM of H₂O₂, but the %viability decreased with H₂O₂ of 3 μM and above. The LC₅₀ value of H₂O₂ for hippocampal neurons was calculated to be 2.46 μM, and the %viability decreased to 16.49% with 10 μM of H₂O₂ (Fig. 3b). The LC₅₀ value of H₂O₂ has previously been reported to be about 40 μM at the cell density of 700 cells/mm² and about 92 μM at that of 2000 cells/mm², after 24 h of incubation, for cortical neurons.²¹ In comparison, we obtained a lower LC₅₀ value (2.46 μM), which might be caused by the difference in cell type and/or the lower cell density (100 cells/mm²) used for our system.

H₂O₂ is known as a trigger of oxidative stress-induced apoptosis.^16,21 Accordingly, we observed the round neuronal morphology of neurons with no or few neurites, one of the major features of apoptosis, after the treatment of H₂O₂. In addition, the weak and broken degenerative neurites and cellular debris after degeneration in the CLSM images also suggested H₂O₂-induced apoptosis.^37,38

CBD is known to be an antioxidant,³⁹ with the similar oxidation potential to butylhydroxytoluene (BHT).^9,40 CBD was reported to reduce the toxic levels of glutamate-induced neurotoxicity to about 40%, with its neuroprotective ability that was similar to BHT and more efficient than commercial antioxidants, such as ascorbate and α-tocopherol, for primary cortical neurons.⁹ Phenolic cannabinoids, including CBD, also have been shown to be the potent cannabinoid receptor 1-independent antioxidants, and protected the mouse hippocampal HT22 cell line up to ∼60% from oxidative stress induced by H₂O₂ ([CBD]=5 μM).¹⁷

In addition, CBD (10 μM) was reported to reduce the death of neuron-like PC12 cells from 39% to 12% against β-amyloid peptides, and decrease the reactive oxygen species production, lipid peroxidation, intracellular calcium, and caspase-3 levels.⁴¹ Several other compounds have also been studied for potential protection of cortical or hippocampal neurons against H₂O₂.^42–44 Y-27632, a Rho-associated kinase (ROCK) inhibitor, increased the cell viability to 87% from 66.5% against H₂O₂ at maximum.⁴² N-Acetyl cysteine, inhibiting the oxidative stress by downregulating mitogen-activated protein kinase (MAPK) signal transduction and tau phosphorylation, was reported to be effective in the neuroprotection against H₂O₂.⁴³ In the case of vitamin E, a fat-soluble antioxidant, also exhibited a neuroprotective capability, increasing the cell viability from 32% to 78% in the hippocampal neuron culture.⁴⁴

In this work, we investigated the neuroprotective effect of CBD against H₂O₂ for primary hippocampal neurons. Specifically, we used 10 μM of H₂O₂ based on our result of the neurotoxicity effect of H₂O₂: this concentration of H₂O₂, inducing about 80% cell death, gave a suitable condition for neuroprotective studies. We also limited the CBD concentrations to less than 10 μM, considering the previous studies on the plasma and brain pharmacokinetic profile of CBD in rats and mice, indicating that the CBD concentrations of ≥10–20 μM in the in vitro studies would be supraphysiological.^45,46

The CLSM images clearly showed that CBD was effective in the neuroprotection of primary hippocampal neurons against H₂O₂ (10 μM) (Fig. 4a). The quantitative analysis on the neuroprotection was performed by calculating the protection ratio of CBD, PR_[CBD], by dividing the %viability values for [+H₂O₂][+CBD] cases by that for the [+H₂O₂][−CBD] case ([H₂O₂]=10 μM).

The bar graph of PR_[CBD] versus [CBD] confirmed the concentration-dependent neuroprotective effects of CBD against H₂O₂ in the in vitro culture of primary hippocampal neurons (Fig. 4b). For example, the PR_[CBD] value was 1.88 for 3 μM of CBD, which further increased to 2.40 for 5 μM of CBD. Interestingly, this PR_[CBD] value was similar to the reported maximum PR value for vitamin E, 2.43,⁴⁴ implying that the antioxidant property of CBD played an important role in the neuroprotection against H₂O₂. The preincubation of the neurons with CBD (5 μM, 1 h) did not lead to any noticeable neuroprotection against H₂O₂.

Although CBD increased the %viability against H₂O₂, showing its neuroprotective capability, the analysis of the high-resolution CLSM images indicated that CBD did not completely prevent the neurite degeneration in apoptotic process even at its concentration of 5 μM (Fig. 5). Therefore, we hypothesized that CBD acted as an antioxidant based on its phenolic structure and partially protected the hippocampal neurons from the initiation and progress of apoptosis caused by oxidative stress. It has been reported that CBD protected oligodendrocyte progenitor cells from apoptosis by decreasing the expression of the proapoptotic proteins or apoptotic effectors, such as Bax and caspase-12, and also increasing the antiapoptotic proteins, such as Bcl-2.¹⁸

Another possibility would be the regulation of [Ca²⁺]_i.^10–12 In the H₂O₂-induced apoptosis was reported the [Ca²⁺]_i increase, by the disruption of Ca²⁺-storing organelles and also calcium influx through activation of several receptors and channels, such as N-methyl-D-aspartate (NMDA) subtype receptor and transient receptor potential melastatin 7 (TRPM7) channel.^21,23–25 Previous reports also showed that the H₂O₂-induced cell death could be prevented by controlling the Ca²⁺ influx. For example, H₂O₂-induced neuronal apoptosis was inhibited by using the antagonist of NMDA receptors, such as MK801.²⁵ The cytotoxicity of H₂O₂ was also attenuated by blocking the TRPM7 channel with the antagonist, 2-APB, or knocking down the TRPM7 channel expression.²⁴

Another plausible mechanism on the neuroprotective effects of CBD against H₂O₂ might be activation of selective receptor mechanisms; H₂O₂ is reported to reversibly sulfenylate cysteines C201 and C208 and deactivate monoacylglycerol lipase, which cleaves 2-arachidonoyl-sn-glycerol, a ligand for cannabinoid receptor type 1 receptor.⁴⁷